Test for Detecting Xanthomonas axonopodis pv. allii

ABSTRACT

The present invention relates to novel tools for detecting  Xanthomonas axonopodis  pv. allii, in particular the molecular detection of specific polynucleotide sequences of said strain.

The present invention relates to novel tools for detecting Xanthomonasaxonopodis pv.

Xanthomonas axonopodis pv. allii is the agent responsible for bacterialblight of onion, one of the major health constraints in the cultivationof Alliaceae. This disease was observed for the first time in 1971 inBarbados (West Indies), then in 1975 in several islands of the Hawaiianarchipelago. In the 1980s, this bacterium was found in Brazil, in Cubaand in Mauritius. Between 1990 and 2000, it reached the United States,Venezuela, South Africa and Japan, causing considerable damage.Xanthomonas axonopodis pv. allii was characterized for the first time inHawaii in 1978 (Alvarez et al., Physiopathology; 68: 1132-1136, 1978).

Xanthomonas axonopodis pv. allii is pathogenic for Alliaceae, inparticular garlic, leek, chive shallot and onion (Roumagnac et al., Int.J. Syst. Evol. Microbiol., 54: 15-24, 2004), the disease tending to bemore severe in onion. This bacterium has also proven to be pathogenicfor species of the Citrus genus during experimental infections byinfiltration (Gent et al., Phytopathology, 95(8): 918-925, 2005); thereis, however, no evidence showing that this bacterium is pathogenic inthe field for the Citrus genus.

In onion, Xanthomonas axonopodis pv. allii causes lesions on the aerialtissues of the plant (leaves and floral scapes), characterized by thepresence of small lenticular watersoaked spots which spread and progressrapidly into chlorosis then necrosis. The disease is promoted by hightemperatures (above 27° C.) and large foci occur rapidly (7 to 10 days)after a period of humid and rainy weather. Because of the reduction infoliage, the plants shrivel up and the bulbs diminish, thereby leadingto considerable yield losses, of about from 10 to 50%. For example, inthe United States, yield losses of 20% or more are commonly observed ininfected fields.

It has been shown that the bacterium can be present in the onion seeds(Roumagnac et al., Eur. J. Plant Pathol., 106: 867-877, 2000), that thedisease can spread on a large scale, in particular through commercialseed exchanges, and that 4 infected seeds out of 10 000 is sufficient totrigger an epidemic (Roumagnac et al., Phytopathology, 94: 138-146,2004).

Within crops, wind and irrigation, and in particular irrigation throughsprinklers, can provide greater dissemination of the disease, as is alsothe case with storms and hail. In addition, the bacterium is capable ofsurviving on crop debris which is then disseminated by adhering toworkers' clothing and to equipment.

Owing to the size of the epidemics and the small proportion ofcontaminated seeds necessary to trigger sizeable epidemics, thisbacterium was placed on the alert list of the European Plant ProtectionOrganization (EPPO) in 2006. A procedure is ongoing for it to beincluded in the A1 list of EPPO quarantine organisms.

Measures for combating bacterial blight of onion are available, forexample the use of healthy seeds, the destruction of self-sown onions,the destruction of plant debris, rotations, chemical control. Inaddition, international marketing and exchange of seeds are the mainagents for the spread of this disease and represent a risk ofintroduction of the pathogenic agent into healthy regions or ofintroduction of new genotypes into regions already contaminated. It istherefore necessary to be able to guarantee the good health quality ofthese seeds. To do this, rapid, reliable and sensitive diagnostic testswhich make it possible to routinely certify Allium seed lots areessential. At the current time, such diagnostic tools do not exist. Theidentification methods currently used are based on isolating thebacterium from the infected plant material and culturing it on asuitable medium, in particular NCTM1 medium (Roumagnac et al., Eur. J.Plant Pathol., 106: 867-877, 2000). The isolation of the bacterium takesseveral days, and the identity of the bacterium must then be verified bybiochemical methods, molecular typing and/or pathogenicity tests (Gentet al., Phytopathology, 94(2): 184-195, 2004; Picard et al.,Phytopathology, 98(9): 919-925, 2008). Furthermore, the presence ofother bacteria in the samples can in certain cases reduce thesensitivity of the test and cause the detection of false positives.

A test for identification by PCR amplification of a specific sequence ofXanthomonas axonopodis pv. allii has been developed (Humeau et al.,Phytopathology, 96(12): 1345-1354, 2006; Picard et al., Phytopathology,98(9): 919-925, 2008). This test consists in carrying out a nested PCRusing, in a first amplification step, the primers PXaa1U (SEQ ID NO:1)/PXaa1L (SEQ ID NO: 2), then, in a second step, the primers NXaa1U(SEQ ID NO: 3)/NXaa1L (SEQ ID NO: 4). However, although this method issensitive and reliable, it does not allow the detection of all strains,in particular those originating from Barbados and Brazil. The fault indetection of certain strains would be explained by the strongintra-pathovar genetic diversity in Xanthomonas axonopodis pv. allii(Picard et al., Phytopathology, 98(9): 919-925, 2008; Gent et al.,Phytopathology, 94(2): 184-195, 2004; Gent et al., Phytopathology,95(8): 918-925, 2005). In addition, a study of genetic diversity hasshown that the polymorphism of the strains is variable depending ontheir geographic origin: there are strong-diversity regions (inparticular the United States, South Africa) and weak-diversity regions(for example, Venezuela, Hawaii, Barbados).

Consequently, since no test is today available for detecting all of theXanthomonas axonopodis pv. allii strains, the development of a reliable,sensitive diagnostic tool which allows the detection of all the strains,regardless of their geographic origin, is essential in order to optimizethe combating of diseases caused by this bacterium and to improve healthmonitoring during international exchanges.

The inventors have now identified two target DNA sequences specific forXanthomonas axonopodis pv. allii. These target sequences, called “PIL”and “AVR”, are represented in the appended sequence listing undernumbers SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The PIL marker,demonstrated by the Random Amplification of

Polymorphic DNA, or RAPD, technique, is present in approximately 70% ofXanthomonas axonopodis pv. allii strains. It has been found that theprimers PXaa1U (SEQ ID NO: 1)/PXaa1L (SEQ ID NO: 2) and NXaa1U (SEQ IDNO: 3)/NXaa1L (SEQ ID NO: 4) used by Humeau et al. (Phytopathology, 96:1345-1354, 2006) hybridize to this target sequence.

The “AVR” marker, identified by the AFLP (for Amplified Fragment LengthPolymorphism) method, is at least present in all the strains which donot have the “PIL” marker.

Thus, owing to the complementarity of these markers, the joint searchfor said markers allows selective diagnosis of all the known strains ofXanthomonas axonopodis pv.

A subject of the present invention is therefore an isolatedpolynucleotide which can be obtained from Xanthomonas axonopodis pv.allii, characterized in that it is chosen from the group made up of:

a) the polynucleotide of sequence SEQ ID NO: 6;

b) a polynucleotide having 85%, preferably 90%, and more preferentially95% identity with the sequence SEQ ID NO: 6;

c) any fragment of at least 15 bp of said polynucleotide;

d) any polynucleotide complementary to one of the polynucleotides a) orb);

e) any polynucleotide capable of selectively hybridizing, understringent conditions, with one of the polynucleotides a), b), c) or d).

The identity percentages to which reference is made in the context ofthe disclosure of the present invention are determined on an overallalignment of the sequences to be compared, using the Needleman andWunsch algorithm (J. Mol. Biol. 48, 443-453, 1970). This sequencecomparison can be carried out, for example, using the “Geneious”alignment software (Drummond A J, Ashton B, Cheung M, Heled J, Kearse M,Moir R, Stones-Havas S, Thierer T, Wilson A; 2009, Geneious v4.7).

Stringent hybridization conditions, for a given polynucleotide, can beidentified by those skilled in the art according to the size and thebase composition of the polynucleotide concerned, and also to thecomposition of the hybridization mixture (in particular, pH and ionicstrength). Generally, stringent conditions, for a polynucleotide ofgiven size and given sequence, are obtained by working at a temperatureapproximately 5° C. to 10° C. below the melting temperature (Tm) of thehybrid formed, in the same reaction mixture, by this polynucleotide andthe polynucleotide complementary thereto.

The present invention encompasses in particular polynucleotides whichcan be used as amplification primers for obtaining a nucleic acidsequence in accordance with the invention, or as nucleic acid probes fordetecting said sequence. It encompasses in particular the pairs ofprimers which can be used for amplifying a polynucleotide of sequenceSEQ ID NO: 6, or a fragment thereof.

By way of nonlimiting examples of pairs of primers in accordance withthe invention, mention will in particular be made of:

the pair of primers made up of the polynucleotides PXaa2U (SEQ ID NO: 7)and PXaa2L (SEQ ID NO: 8), which allows the amplification of a 995 bpfragment of the sequence SEQ ID NO: 6;

the pair of primers made up of the polynucleotides NXaa2U (SEQ ID NO: 9)and NXaa2L (SEQ ID NO: 10), which allows the amplification of a 401 bpfragment of the sequence SEQ ID NO: 6.

FIG. 1 represents the sequence SEQ ID NO: 6. The sequence fragments inbold delimit the primers PXaa2U, PXaa2L, NXaa2U and NXaa2L.

Another subject of the present invention is the use of nucleic acidmolecules in accordance with the invention for screening for Xanthomonasaxonopodis pv. allii, and in particular the strains which do notcomprise the “PIL” marker.

A subject of the present invention is also a method of screening forXanthomonas axonopodis pv. allii, characterized in that it comprises:

-   bringing DNA of a biological sample that may contain said bacterium    into contact with one or more polynucleotide(s) in accordance with    the invention, under conditions which allow selective hybridization    between said polynucleotide(s) and the target sequence SEQ ID NO: 6,    if said sequence is present in said DNA;-   detecting said hybridization.

The biological sample used may be either a culture of bacteria isolatedfrom the plant or from the seeds of Alliaceae on which it is desired tocarry out the detection, or directly the potentially infected seeds ofAlliaceae, or a sample of the potentially infected plant.Advantageously, the detection is carried out on onion seeds.

The detection of the hybridization can be carried out by any means knownin themselves to those skilled in the art. When a polynucleotide inaccordance with the invention, labeled beforehand with a suitable label,is used as nucleic acid probe, the hybridization of the target sequencewith the labeled probe is detected directly.

When two polynucleotides, constituting a pair of primers in accordancewith the invention, are used, the hybridization of the primers with thetarget sequence is demonstrated by means of a polymerase chain reaction(PCR) amplification, and then by detection of the amplification product,in particular on agarose gel.

In this case, the screening method in accordance with the inventioncomprises at least the following steps:

-   i) bringing the DNA of a biological sample to be tested into contact    with a pair of primers in accordance with the invention, under    conditions which allow selective hybridization between the primers    and the target sequence SEQ ID NO: 6;-   ii) carrying out a polymerase chain reaction amplification under    conditions which allow the amplification of the target sequence SEQ    ID NO: 6;-   iii) detecting the amplification product.

Preferably, the detection is carried out by nested PCR. In thisembodiment of the invention, the screening method in accordance with theinvention also comprises a second PCR amplification step (step iv)carried out using a second pair of primers allowing the amplification ofan internal fragment of the amplification product from the first step.

By way of example, the first amplification step (step ii) of the methodin accordance with this embodiment can be carried out using the pair ofprimers made up of the polynucleotides PXaa2U (SEQ ID NO: 7) and PXaa2L(SEQ ID NO: 8), and the second amplification step (step iv) using thepair of primers made up of the polynucleotides NXaa2U (SEQ ID NO: 9) andNXaa2L (SEQ ID NO: 10).

In order to be able to detect all the Xanthomonas axonopodis pv. alliistrains, i.e. the strains which contain the “PIL” and “AVR” markers, andthose which contain only one of these two markers, another particularlyadvantageous embodiment of the present method allows the detection ofthe target sequence SEQ ID NO: 5 (“PIL” marker) in addition to that ofSEQ ID NO: 6 (“AVR” marker).

In this particular embodiment, one or more polynucleotide(s) inaccordance with the invention capable of selectively hybridizing withthe target sequence SEQ ID NO: 6, and one or more polynucleotide(s)capable of selectively hybridizing, under stringent conditions, with thetarget sequence SEQ ID NO: 5, are used.

When the method of detection according to the invention comprises a PCRamplification step, pairs of primers which allow the amplification of apolynucleotide of sequence SEQ ID NO: 5, or of a fragment thereof, andprimers in accordance with the invention which allow the amplificationof a polynucleotide of sequence SEQ ID NO: 6, or of a fragment thereof,are used.

In this case, in step i) of the method in accordance with the invention,the DNA of the sample is also brought into contact with one or morepolynucleotide(s) capable of selectively hybridizing, under stringentconditions, with the target sequence SEQ ID NO: 5, and the conditions ofstep ii) allow, in addition to the amplification of the target sequenceSEQ ID NO: 6, the amplification of the target sequence SEQ ID NO: 5.

Preferably, the pairs of primers that can be used for amplifying thepolynucleotide of sequence SEQ ID NO: 5 are:

the pair of primers PXaa1U (SEQ ID NO: 1) and PXaa1L (SEQ ID NO: 2),which allows the amplification of a 694 bp fragment of the sequence SEQID NO: 5; and

the pair of primers NXaa1U (SEQ ID NO: 3) and NXaa1L (SEQ ID NO: 4),which allows the amplification of a 444 bp fragment of the sequence SEQID NO: 5.

FIG. 2 represents the sequence SEQ ID NO: 5. The sequence fragments inbold delimit the primers PXaa1U, PXaa1L, NXaa1U and NXaa1L. When it is aquestion of a method of detection according to the invention comprisingtwo amplification steps (“nested PCR” embodiment), a pair of primersallowing the amplification of all or part of the polynucleotide ofsequence SEQ ID NO: 5 is used in the first amplification step (stepii)), in addition to the pair of primers according to the inventionallowing the amplification of all or part of the polynucleotide ofsequence SEQ ID NO: 6. In the second PCR amplification step (step iv), apair of primers allowing the amplification of an internal fragment ofthe product of amplification of the sequence SEQ ID NO: 5 obtained inthe first step is used, in addition to the primers according to theinvention making it possible to amplify an internal fragment of theproduct of amplification of the sequence SEQ ID NO: 6.

In one particularly advantageous embodiment of the method according tothe invention, the amplification step ii) is carried out with the pairof primers PXaa1U (SEQ ID NO: 1) and PXaa1L (SEQ ID NO: 2) and the pairof primers PXaa2U (SEQ ID NO: 7) and PXaa2L (SEQ ID NO: 8), and theamplification step iv) is carried out with the pair of primers NXaa1U(SEQ ID NO: 3) and NXaa1L (SEQ ID NO: 4) and the pair of primers NXaa7U(SEQ ID NO: 9) and NXaa2L (SEQ ID NO: 10). This embodiment has asensitivity threshold of approximately 1 contaminated seed out of 27300: it is therefore particularly reliable for evaluating the state ofhealth of a sample of seeds, and it can be used for the certification ofAlliaceae seeds, since the rate of seed contamination recorded forepidemics of bacterial blight of onion in the tropical environment is4.5/10 000.

The embodiments of the method according to the invention targeting boththe “PIL” and “AVR” target genes have the advantage of being able todetect all the Xanthomonas axonopodis pv. allii strains, as has beendemonstrated by the inventors on a “worldwide collection”, listed intable I (cf. hereinafter example 2.1), made up of 87 strains originatingfrom various regions of the world, whereas most of the Xanthomonasstrains different than the Xanthomonas axonopodis pv. allii pathovar arenot detected. Only a few strains classified in genetic subgroups 9.1 and9.2 of Xanthomonas axonopodis, which are not pathogenic for onion, arealso detected. However, the latter can be easily distinguished from theXanthomonas axonopodis pv. allii strains by means of a restrictionprofile analysis, for example using the NheI enzyme which cleaves onlythe amplification product of Xanthomonas euvesicatoria strains (group9.2), or using the CfrI enzyme which cleaves only the amplificationproduct of Xanthomonas axonopodis subgroup 9.1, in particularXanthomonas axonopodis pv. begoniae.

A subject of the present invention is also a kit for detectingXanthomonas axonopodis pv. allii, comprising one or morepolynucleotide(s) in accordance with the invention capable ofselectively hybridizing, under stringent conditions, with the targetsequence SEQ ID NO: 6. Preferably, the detection kit also comprises oneor more polynucleotide(s) capable of selectively hybridizing, understringent conditions, the target sequence SEQ ID NO: 5.

The present invention will be understood more clearly by means of thefurther description which follows, which refers to nonlimiting examplesillustrating the implementation of the present invention for detectingXanthomonas axonopodis pv.

EXAMPLE 1 Identification of the “PIL” and “AVR” Markers

1.1. Identification of the “PIL” marker

The strains used in the present study were cultured on YPGA medium (7g.l⁻¹ of yeast extract, 7 g.l⁻¹ of peptone, 7 g.l⁻¹ of glucose, 18 g.l⁻¹of agar, pH 7.2) or on modified Wilbrink medium (10 g.l⁻¹ sucrose, 5g.l⁻¹ bactopeptone, 5 g.l⁻¹ of yeast extract, 0.5 g.l⁻¹ of K₂HPO₄, 0.25g.l⁻¹ of MgSO₄/7H₂O, 0.05 g.l⁻¹ of Na₂SO₃, 15 g.l⁻¹ of agar, pH 6.9-7;Ron et al., Agron. Trop.; 43: 244-251, 1988) for the strains showingweak growth on the YPGA medium.

Random amplification of polymorphic DNA, or RAPD, was then carried out.For this, the total DNA of twenty-two Xanthomonas axonopodis pv. alliistrains originating from various geographic regions (cf. below, table I,the strains labeled with “b”) and of six other Xanthomonas (cf. below,table II, the strains labeled was was extracted from 2 ml of bacterialculture using the “DNeasy blood and tissue” kit (Qiagen, Courtaboeuf,France) according to the supplier's instructions.

A PCR amplification was carried out on the DNA extracts resulting fromeach strain. The reaction medium of 25 μl contains 25 ng of bacterialgenomic DNA, 3 mM of MgCl₂, 0.4 μM of primers, 2.5 units of Taq DNApolymerase (Invitrogen, Merelbeke, Belgium), 100 μM of each of the dNTPs(Roche Diagnotics, Meylan, France) in 20 mM Tris-HCl medium and 50 mM ofKCl buffer (pH 8.4). About a hundred short primers of 10 nucleotides ofarbitrarily defined sequence, originating from the 80, 70 and 60% GCkits (Genome Express, Meylan, France), were tested.

The amplification was carried out under the following conditions:

initial denaturation at 94° C. for 7 min,

40 amplification cycles each comprising 3 phases: 94° C. for 1 min(denaturation), 35° C. for 1 min (hybridization of the primers on theDNA) and 72° C. for 2 min (polymerization),

extension at 72° C. for 5 min.

The amplification products were subjected to 2% NuSieve agarose gelelectrophoresis and visualized by fluorescence after staining withethidium bromide.

It was found that none of the fragments was present in all of theXanthomonas axonopodis pv. allii strains tested and absent from theother pathovars. Among the amplified fragments found in most of theXanthomonas axonopodis pv. allii strains and absent from the otherstrains, the fragment amplified with the primer called 80-21 (SEQ ID NO:11; 5′ ACGCGCCAGG 3′), of approximately 940 bp, is present in 70% of theXanthomonas axonopodis pv. allii strains tested.

This fragment of interest was excised from the agarose gel for theXanthomonas axonopodis pv. allii strains CFBP6364, CFBP6366, CFBP6369and CFBP6379, extracted using the “QIAquick gel Extraction” extractionkit (Qiagen, Courtaboeuf, France), and then cloned by ligation into thepGEM-T Easy vector according to the supplier's instructions (Promega,Madison, USA). The sequence was then determined by sequencing using theT7 and SP6 primers, which hybridize to the regions bordering the insertcloned into the pGEM-T Easy vector. This fragment is 99% identicalbetween these four strains, and a search in the GenBank database revealsthat it shares 97% identity with two contiguous portions (respectivelyof 828 and 102 bp) belonging to the PILW and PILX genes of Xanthomonaseuvesicatoria, which genes encode pilus assembly proteins.

It should be noted that none of the other fragments identified by RAPDwere present in the strains of which the genome was not amplified by the80-21 primer.

1.2. Identification of the “AVR” Marker

The Xanthomonas axonopodis pv. allii strains CFBP6369, CFBP6380,CFBP6384, JX36-1, CFBP6386 and CFBP6382, representative of the strainsnot amplified by the 80-21 primer, were analyzed using the AFLPtechnique. A Xanthomonas citri pv. citri strain (IAPAR 306) and aXanthomonas campestris pv. campestris strain (CFBP 5251) were used ascontrol.

The AFLP analysis was carried out as described by Ah-You et al.(International Journal of Systematic and Evolutionary Microbiology, 59:306-318, 2009).

The bacterial DNA, extracted as previously indicated, was digestedjointly with SacI and MspI, and then ligation with the SacI and MspIlinkers was carried out for 3 hours at 37° C. by adding 2.5 μl ofdigestion product to 22.5 μl of a ligation mixture containing 2 μM ofthe MspI linker, 0.2 μM of the SacI linker (Applied Biosystems,Courtaboeuf, France) and 2 units of T4 DNA ligase (New England BiolabsOzyme) in 1× ligation buffer. A PCR preamplification is carried out in15 μl of reaction mixture consisting of 5 μl of the ligation mixturediluted to 1/10th, 2.5 mM of MgCl₂, 0.23 μM of each of the primers PSAC(SEQ ID NO: 12) and PMSP (SEQ ID NO: 13), 0.45 μM of each dNTP, and 0.5unit of Taq DNA polymerase (Goldstar Red, Eurogentec, Seraing, Belgium)in a 1× Goldstar buffer. The amplification was carried out under thefollowing conditions:

initial extension at 72° C. for 2 min,

initial denaturation at 92° C. for 2 min,

25 amplification cycles, each comprising 3 phases: 94° C. for 30 sec(denaturation), 56° C. for 30 sec (hybridization of the primers on theDNA) and 72° C. for 2 min (polymerization),

extension at 72° C. for 10 min.

The amplification products are then diluted 10-fold in HPLC water,before the selective amplification. The selective amplification iscarried out using the pairs of unlabeled primers MspI (SEQ ID NO: 14)+A, T, G or C and Sad C (SEQ ID NO: 15), or MspI (SEQ ID NO: 14) +A, T,G or C and SacI+CT (SEQ ID NO: 16). The amplification conditions are thesame as those described for the preamplification, with the exception ofthe concentration of the primers SacI+C and SacI+CT, which is 0.12 μM.

The amplification products were then analyzed on a 5% acrylamide gel(Risterucci et al., Theor. Appl. Genet.; 101: 948-955, 2000) and thenthe DNA was visualized by silver staining (Qu et al., Electrophoresis;26: 99-101, 2005). Owing to the complexity of the AFLP profiles obtainedusing two selective bases in the amplification step, only the profilesobtained with three selective bases were exploited for recovering thefragments of interest.

Two fragments present in the majority of the Xanthomonas axonopodis pv.allii strains, including the strains which are not amplified by the80-21 primer, were visualized.

In order to identify these fragments, the DNA was extracted from theacrylamide gel in 50 μl of buffer. 5 μl were then subjected to selectiveamplification, as described by Ah-You et al. (International Journal ofSystematic and Evolutionary Microbiology, 59: 306-318, 2009), using thepairs of primers MspI (SEQ ID NO: 14)+A or T and SacI+CT (SEQ ID NO:16). The amplification conditions are the same as those described forthe preamplification, with the exception of the concentration of theprimer SacI+CT, which is 0.12 μM.

The amplified fragments were then introduced by ligation into the pGEM-TEasy vector as described above, and then E. coli bacteria weretransformed with the vectors containing the inserts. Despite the morestringent conditions for amplification with three selective bases, thesequencing of the clones obtained revealed that the DNAs had aheterogeneous composition, which is no doubt explained by a comigrationof amplification products of similar size or a contamination during theexcision step. Nevertheless, using the amplification with threeselective nucleotides, an interesting clone was obtained from the AFLPfragments originating from the CFBP 6382 strain. Sequencing of theinsert of this clone followed by a search in the GenBank databaserevealed the presence of a 270 bp sequence having 90% identity with aportion of the avrRxv avirulence gene of Xanthomonas euvesicatoria(GenBank accession number L20423). On the basis of this sequence, theprimers F1154 (SEQ ID NO: 17) and R1391 (SEQ ID NO: 18) were generatedand then a PCR was carried out using these primers on the strainsCFBP6366, CFBP6380, CFBP6384, JX36-1, CFBP 6386, CFBP6357 and CFBP6382.

The PCR amplification was carried out in 25 μl of reaction mixtureconsisting of 20 ng of total DNA, 3 mM of MgCl₂, 0.2 μM of primers, 1.25units of Dap Goldstar polymerase (Eurogentec, Seraing, Belgium), and 100μM of each of the dNTPs

(Roche Diagnotics, Meylan, France). The following amplificationconditions were used:

initial extension at 72° C. for 2 min,

initial denaturation at 94° C. for 5 min,

40 amplification cycles, each comprising 3 phases: 95° C. for 1 min(denaturation), 60° C. for 1 min (hybridization of the primers on theDNA) and 72° C. for 2 min (polymerization),

extension at 72° C. for 5 min.

An amplicon of 238 bp was obtained for each of the strains tested:depending on the strains, the 238 bp fragment shared 90 to 99% of theavrRxv avirulence gene of Xanthomonas euvesicatoria (GenBank accessionnumber L20423). In order to obtain larger DNA fragments so as to designPCR primers specific for Xanthomonas axonopodis pv. allii,amplifications were carried out using the pair of primers F202 (SEQ IDNO: 19), corresponding to nucleotides 202-219 of the avrRxv avirulencegene of Xanthomonas euvesicatoria (GenBank accession number L20423) andR1391 (SEQ ID NO: 18) chosen in the 238 bp amplicon, and also the pairof primers F 1154 (SEQ ID NO: 17) and R2149 corresponding to nucleotides2131-2149 (SEQ ID NO: 20) of the avrRxv avirulence gene of Xanthomonaseuvesicatoria (GenBank accession number L20423). A fragment of 1948 bpwas amplified for the strains CFBP6366, CFBP6386, CFBP6357 and CFBP6382,a fragment of 1950 bp for the strains CFBP 6384 and JX36-1, and apartial sequence of 1504 bp for the strain CFBP 6380. All thesesequences exhibit between 87 and 98% identity with the avrRxv gene ofXanthomonas euvesicatoria.

EXAMPLE 2 Specific Detection of Xanthomonas axonopodis pv. allii 2.1.Specificity of the Detection by Multiplex Nested PCR

Pairs of primers were designed, on the basis of the “PIL” (SEQ ID NO: 5)and “AVR” (SEQ ID NO: 6) markers, by selecting the most conservedregions of these two markers among the Xanthomonas axonopodis pv. alliistrains.

For the “PIL” marker, the two pairs of primers chosen are the pair ofprimers PXaa1U (SEQ ID NO: 1) and PXaa1L (SEQ ID NO: 2), and the pair ofprimers NXaa1U (SEQ ID NO: 3) and NXaa1L (SEQ ID NO: 4).

For the “AVR” marker, the two pairs of primers chosen are the pair ofprimers PXaa2U (SEQ ID NO: 7) and PXaa2L (SEQ ID NO: 8), and the pair ofprimers NXaa2U (SEQ ID NO: 9) and NXaa2L (SEQ ID NO: 10).

An analysis by multiplex nested PCR was then carried out using thesefour pairs of primers on 87 Xanthomonas axonopodis pv. allii strains(cf. table I hereinafter), 101 phytopathogenic bacterial strains (otherpathovars and species of Xanthomonas, other bacterial genera), and 18saprophytic bacteria from onion (cf. table II hereinafter).

Various types of samples were tested, in particular purified bacterialgenomic DNA, bacterial suspensions and seeds mixed with a bacterialstrain.

The first step of the analysis by multiplex nested PCR was carried outin a reaction mixture of 25 μl consisting of 0.5 to 5 μl of test sample(depending on the type of sample), 3 mM of MgCl₂, 0.2 μM of each of theprimers constituting the pairs of primers PXaa2U (SEQ ID NO: 7)/PXaa2L(SEQ ID NO: 8) and PXaa1U (SEQ ID NO: 1)/PXaa1L (SEQ ID NO: 2), 100 μMof each dNTP, 1.25 units of Taq DNA polymerase (Goldstar Red,Eurogentec, Seraing, Belgium), in a 75 mM Tris-HCl buffer containing 20mM (NH₄)₂SO₄ and 0.01% Tween 20 (pH 8.8).

The amplification conditions used were the following:

initial denaturation at 94° C. for 5 min,

40 amplification cycles, each comprising 3 phases: 95° C. for 1 min(denaturation), 63° C. for 1 min (hybridization of the primers on theDNA) and 72° C. for 2 min (polymerization),

extension at 72° C. for 5 min.

The second step of the multiplex nested PCR was carried out with 1 to 5μl of the amplicons obtained in the first amplification step, underconditions analogous to those described for the first amplificationstep, with the exception of the pairs of primers, which are thefollowing: NXaa7U (SEQ ID NO: 9)/NXaa2L (SEQ ID NO: 10) and NXaa1U (SEQID NO: 3)/NXaa1L (SEQ ID NO: 4) were used. The amplification program wasthe following:

initial denaturation at 95° C. for 5 min,

30 amplification cycles, each comprising 3 phases: 94° C. for 30 sec(denaturation), 57° C. for 30 sec (hybridization of the primers on theDNA) and 72° C. for 40 sec (polymerization),

extension at 72° C. for 5 min.

The amplification products were then analyzed on a 1% agarose gel(amplicon originating from the first amplification step) or a 3% agarosegel (amplicon originating from the second amplification step).

Among the 87 Xanthomonas axonopodis pv. allii strains tested, threetypes of results are obtained during the first amplification step:

i) two amplicons having the expected sizes, 694 bp (a portion of the“PIL” marker) and 995 bp (a portion of the “AVR” marker), were amplifiedfrom all the strains from Brazil, Cuba, Japan, Mauritius, Reunion Islandand Hawaii;

ii) a single amplicon of 694 bp (a portion of the “PIL” marker) wasamplified from all the strains originating from Georgia and fromVenezuela, from four strains from Colorado and from two strainsoriginating from South Africa;

iii) a single amplicon of 995 bp (a portion of the “AVR” marker) wasamplified from all the strains originating from Barbados and from Texas,three strains originating from South Africa and one originating fromColorado.

During the second amplification step, the following fragments, resultingfrom the internal amplification of the amplification products from thefirst step, were observed: two fragments of 444 bp (an internal portionof the “PIL” amplicon) and 401 bp (an internal portion of the “AVR”amplicon) were obtained for the group exhibiting profile i), and asingle amplicon of 444 and 401 bp, respectively, was obtained for groupsii) and iii).

The results obtained for the 87 Xanthomonas axonopodis pv. allii strainstested are summarized in table I below.

In addition, the pathogenicity of these 87 strains was confirmed byverification of Koch's postulates using, for each strain, the hostspecies from which it originates.

TABLE I Response in multiplex nested PCR

694 bp/444 bp 995 bp/401 bp amplicons amplicons Strains

Origin Host (PIL marker) (AVR marker) CFBP 6384

, JX36-1

, CFBP South Africa A. cepa L −/− +/+ 6386

CFBP 6385

, JX36-2

South Africa A. cepa L +/+ −/− CFBP 6367

, JR512

, JR513

, JR514, Barbados A. cepa L −/− +/+ JR515, CFBP 6368

CFBP 6362

, CFBP 6363

, JV593

, Brazil A. cepa L +/+ +/+ CFBP 6377

CFBP 6378

Brazil A. cepa L −/− +/+ CFBP 6364

, CFBP 6365

Cuba A. sativum L +/+ +/+ CFBP 6107*

, CFBP 6108

Japan A. fistulosum L +/+ +/+ JR649, JR650, JR651, CFBP 6374

, Mauritins A. cepa L +/+ +/+ JR653, JR654-1, CFBP 6374, JR653, JR654-1,JR655, CFBP 6376

, JS959, JS960, JS961, JS962 JQ740-1, LMG 16528

, CFBP Reunion Island A. cepa L +/+ +/+ 6366

, CFBP 6369

, CFBP 6357

, JQ759, JR520-1, JR523-1, CFBP 6371

, CFBP 6372, CFBP 6373, CFBP 6375 CFBP 6358

, LA261-18, JX366-2 Reunion Island A. sativum L +/+ +/+ JX-373-2,LB239-18 Reunion Island A. porrum L +/+ +/+ JY317

, CFBP 6379

, JY319

Colorado (USA) +/+ −/− JY320

CFBP 6380

Colorado (USA) A. cepa L −/− +/+ JY274, JY275, JY276

Georgia (USA) +/+ −/− LMG 577, LMG 578, LMG 579

, Hawaii (USA) A. cepa L +/+ +/+ CFBP 6359

, LMG 943, LMG 9487, LMG 9488, LMG 9489, CFBP 6360

, LMG 9491, LMG 9492, CFBP 6361

, LMG 9494 JW200, CFBP 6381

, JW202, CFBP Texas (USA) A. cepa L −/− +/+ 6382

, JW204, JW205, CFBP 6383

CFBP 6387

, JX722

, JX724, JX725, Venezuela A. cepa L +/+ −/− JX726, CFBP 6388

, JX728 ^(a)CFBP, French Collection of Phytopathogenic Bacteria, PlantPathology Station, Angers, France. BCCMTM/LMG, Bacteria collection,Laboratory of Microbiology, Ghent University, Belgium; the other strainsbelong to our laboratory collection (3P, Reunion, France). All thestrains listed in this table are pathogenic for onion. ^(b)Strains usedto evaluate the RAPD markers (22 strains). ^(c)Strains for which theRAPD marker 80-21 was cloned and sequenced (4 strains). ^(d)Strains usedfor evaluating the AFLP markers (6 strains). ^(e)Strains for which theAFLP fragment of 238 bp was cloned and sequenced (7 strains). ^(f)+,fragment of expected size amplified; −, no detectable amplicon.^(g)Strains for which the DNA was amplified by the primers PXaa1U andPXaa1L and sequenced (27 strains). ^(h)Strains for which the DNA wasamplified by the primers PXaa2U and PXaa2L and sequenced (28 strains).^(i)Strains used for the restriction analysis with Cfrl (8 strains).^(j)Strains used for the restriction analysis with NheI (10 strains).*Pathotype strain.

indicates data missing or illegible when filed

An example of a multiplex nested PCR result is illustrated in FIG. 3.Lane 11 represents the negative PCR control, lanes 1 to 10 represent theresults obtained for the strains CFBP 6384, CFBP 6385, CFBP 6380, CFBP6379, CFBP 6369, CFBP 6107, JY 276, CFBP 6387, CFBP 6368 and CFBP 6381.Lane M corresponds to the molecular size markers.

During the multiplex nested PCR tests carried out on the saprophyticstrains from onion and on strains belonging to other bacterial genera,no amplification product was observed. Similarly, the majority of theXanthomonas strains are not amplified by multiplex nested PCR. However,it was found that a few Xanthomonas axonopodis strains classified ingenetic subgroup 9.2 sensu Rademaker were also detected. Depending onthe pathovar, the amplicons of the “PIL” marker and/or that of the “AVR”marker are revealed. In addition, the amplicons of the “AVR” marker arealso detected for the strains of Xanthomonas axonopodis pv. begoniae(genetic subgroup 9.1 sensu Rademaker).

The results obtained for the saprophytic strains from onion and thestrains belonging to other bacterial genera tested are summarized intable II below.

TABLE II Response to multiplex nested PCR

694 bp/444 bp 995 bp/401 bp amplicons amplicons Strains

Taxon (PIL marker) (AVR marker) Genetic subgroup 9.2 LMG 667

, LMG 668

, LMG 905

, Xanthomonas

+/+ +/+ LMG 909, LMG 910

, LMG 913

, LMG 914

, LMG 922

, LMG 926

, LMG927

, LMG 929

, LMG 930

, LMG 931

, LMG 932

, LMG 933

, CFBP 5600

JJ238-20, JJ238-26, JJ238-27, JJ238-28 Xanthomonas axonopodis pv. −/−−/−

CFBP 2910

Xanthomonas axonopodis pv. −/− +/+

LMG 497

, LMG 8019 Xanthomonas axonopodis pv. −/− −/− alfalfae LMG 675

Xanthomonas axonopodis pv. −/− −/− cassiae LMG 8048

Xanthomonas axonopodis pv. +/+ −/− cassavae LMG 861

Xanthomonas axonopodis pv. −/− −/− rich

LMG 686

Xanthomonas axonopodis pv. −/− −/− corccanae LMG 691

Xanthomonas axonopodis pv. −/− −/− cyamopsidis LMG 692

Xanthomonas axonopodis pv. −/− +/+ desmodii LMG 693

Xanthomonas axonopodis pv. +/+ −/− desmodiigangeiici LMG 694

Xanthomonas axonopodis pv. −/− −/−

LMG 698

Xanthomonas axonopodis pv. −/− −/− erythrinae LMG 811* Xanthomonasaxonopodis pv. −/− −/− patelii LMG 844

Xanthomonas axonopodis pv. +/+ +/+ phyllanthi LMG 849

Xanthomonas axonopodis pv. −/− −/− poinsettiicola LMG 955

Xanthomonas axonopodis pv. +/+ −/−

NCPPB 938

Xanthomonas axonopodis pv. +/+ +/+ lespadecae Genetic subgroup 9.1 LMG551

, LMG 7303 

*, CFBP Xanthomonas axonopodis pv. +/+ +/+ 2524

bagoniae Genetic subgroup 9.3 LMG 982*, LMG 539 Xanthomonas axonopodispv. −/− −/− axonopodis LMG 901*, LMG 8285 Xanthomonas axonopodis pv. −/−−/− vuscalorum Genetic subgroup 9.4 LMG 8014, JR518-3 Xanthomonasaxonopodis pv. −/− −/− phaseoli CFBP 2603 Xanthomonas axonopodis pv. −/−−/− manthotis LMG 695

Xanthomonas axonopodis pv. −/− −/−

Genetic subgroup 9.5 C 39, JA Z,899  59-1, CFBP

, IAPAR Xanthomonas axonopodis pv. −/− −/− 306 citri LMG 548

Xanthomonas axonopodis pv. −/− −/−

LMG 558

Xanthomonas axonopodis pv. −/− −/− cajoni CFBP 1716

, CFBP 2933 Xanthomonas axonopodis pv. −/− −/−

LMG 761

 LMG 7429 Xanthomonas axonopodis pv. −/− −/−

Genetic subgroup 9.6 LMG 7387 Xanthomonas axonopodis pv. −/− −/− cajoniLMG 8752

Xanthomonas axonopodis pv. −/− −/− vignicola Other species LMG

, CFBP 4925^(†) Xanthomonas 

 pv. −/− −/−

LMG 904, LMG 915, LMG 911

Xanthomonas

−/− −/− CFBP 1156

Xanthomonas

−/− −/− CFBP 1976

Xanthomonas bromi −/− −/− LMG 710, CFBP 2157^(†) Xanthomonas jragariae−/− −/− CFBP 5251^(†) Xanthomonas campesiris pv. −/− −/− campesiris CFBP2528^(†) Xanthomonas arboricoln pv. −/− −/− juglandis CFBP 2532^(†)Xanthomonas aryzae pv.

−/− −/− CFBP 2542^(†) Xanthomonas

−/− −/− CFBP 2543^(†) Xanthomonas rasicola pv. −/− −/−

CFBP 4188^(†) Xanthomonas

−/− −/− CFBP 4641^(†) Xanthomonas

−/− −/− LMG 673^(†) Xanthomonas

−/− −/− CFBP 4644^(†) Xanthomonas melonis −/− −/− CFBP 4643^(†)Xanthomonas pisi −/− −/− CFBP 4690^(†) Xanthomonas codia −/− −/−CFBP4691^(†) Xanthomonas

−/− −/− CFBP 2523^(†) Xanthomonas

−/− −/− LMG 797

Xanthomonas aryzae pv. −/− −/− aryzicola LMG 892

Xanthomonas 

 pv. −/− −/−

LMG 471

Xanthomonas sacchari −/− −/− Other phytopathogenic bacteria LMG 1222^(†)Burkholderia capana −/− −/− CFBP 2094 Pseudomonas 

 pv. −/− −/−

CFBP1670^(†) Pseudomonas savastanol −/− −/− Run 145 Ralstoniasolanacearum −/− −/− phylotype III Run 215 Ralstonia solanacearum −/−−/− phylotype I Run 17 Ralstonia solanacearum −/− −/− phylotype II Run83 Ralstonia solanacearum −/− −/− phylotype IV LMG 1199^(†) Ralstoniaeutropha −/− −/− LMG 2172^(†) Pseudomonas corrugala −/− −/− LMG 5093

Pseudomonas 

 pv. −/− −/− tomato LMG 5942^(†) Ralstonia pickettii −/− −/− LMG1794^(†) Pseudomonas fluorescens −/− −/− LMG 16206

Pseudomonas putida −/− −/− LMG 2162^(†) Pseudomonas cichorii −/− −/− LMG2129 Burkholderia andropogonis −/− −/− LMG 2804^(†)

 chrysanthemi −/− −/− Saprophytic strains isolated from onion JS923,JS924 Ervinia sp. −/− −/− JR593 Burkholderia sp. −/− −/− JR594-1Stenotrophomonas sp. −/− −/− JR594-2 Pseudomonas sp. −/− −/− JR656-3Klebsiella sp. −/− −/− JR656-5 Pantoea sp. −/− −/− JR656-6 Pantoea sp.−/− −/− JS741-1 Burkholderia sp. −/− −/− JS741-2, JS741-4, JS741-12Flavimonas

−/− −/− JS741-5 Klebsiella sp. −/− −/− JS741-6

 agglomerans −/− −/− JS853 Pantoea sp. −/− −/− JS741-14 Enterobacter sp.−/− −/− JS741-15, JS741-16 Pseudomonas aeruginosa −/− −/− ^(a)CFBP,French Collection of Phytopathogenic Bacteria, Plant Pathology Station,Angers, France: BCCMTM/LMG, Bacteria collection, Laboratory ofMicrobiology, Ghent University, Belgium; NCPPB, the National Collectionof Plant Pathogenic Bacteria (NCPPB, CSL, York, United Kingdom); theother strains belong to our laboratory collection (3P, Reunion, France),except for the strain IAPAR 306 which was provided by the IAPAR(Instituto Agronomico do Parana [Agronomic Institute of Parana],Londrina PR, Brazil) and for which the complete genome is available (DaSilva, 2002). All the strains listed in this table are pathogenic foronion. ^(b)Strains used for the tests for pathogenicity on onion (Alliumcepa L., cv. Red Creole). ^(c)+, amplified fragment of the expectedsize; −, no amplicon detected. ^(d)Strains used to evaluate the RAPDmarkers (6 strains). ^(e)Strains for which the DNA was amplified by theprimers PXaa1U and PXaa1L and sequenced (18 strains). ^(f)Strains forwhich the DNA was amplified by the primers PXaa2U and PXaa2L andsequenced (17 strains). ^(g)Strains used for the restriction analysiswith CfrI (3 strains). ^(h)Strains used for the restriction analysiswith NheI (12 strains). ^(†)Type strain. *Pathotype strain.

indicates data missing or illegible when filed

Pathogenicity tests were carried out for all the strains of table II,using the onion cultivar Red Creole (Roumagnac et al., Eur. J. PlantPathol., 106: 867-877, 2000). These tests showed that although they aredetected by multiplex nested PCR, none of the strains that do not belongto the allii pathovar are pathogenic to onion. In addition, said strainscan easily be distinguished from the Xanthomonas axonopodis pv. alliistrain by restriction profile analysis. Indeed, analysis of theamplicons obtained during the second amplification step indicates thatthe NheI restriction enzyme does not cleave the 444 bp fragment(amplicon of the “PIL” marker) originating from the Xanthomonasaxonopodis pv. allii strain, whereas it cleaves the amplificationproduct from the Xanthomonas euvesicatoria strain, generating twofragments of 355 and 89 bp, respectively. In the same way, the CfrIrestriction enzyme generates two fragments of 343 and 58 bp from the 401bp amplification product (amplicon of the “AVR” marker) from Xanthomonasaxonopodis pv. begoniae, whereas it does not cleave the ampliconoriginating from Xanthomonas axonopodis pv.

2.2. Sensitivity of the Detection by Multiplex Nested PCR

The sensitivity of the method of detection by multiplex nested PCR wasdetermined using bacterial suspensions optionally mixed with samples ofonion seeds.

The suspensions of the Xanthomonas axonopodis pv. allii strains CFBP6366, CFBP 6385 and CFBP 6367 were tested. For this, the suspensionscontaining 1×10⁸ cfu.ml⁻¹ were serially diluted 10-fold. Samplescontaining 10 g of healthy onion seeds were placed in 50 ml of sterile0.01 M Sigma 7-9 buffer (pH 7.2) (Sigma, Saint-Quentin Fallavier,France) and were inoculated with bacterial suspensions at a finalconcentration ranging from 1×10¹ cfu.ml⁻¹ to 1×10⁷ cfu.ml⁻¹. Thenegative control was inoculated with buffer. In parallel, the samedilution series not mixed with the seeds were analyzed. After 48 hoursof maceration at 4° C., the samples were firstly inoculated onto NCTM1semi-selective medium (Roumagnac et al., Eur. J. Plant. Pathol.; 106:867-877, 2000), and secondly, the bacterial genome was extractedaccording to the rapid alkaline extraction method of Audy et al.(Phytopathology; 86: 361-366, 1993). 4 ml of each sample werecentrifuged at 10 000 g for 30 min at 4° C. and then the pellet wasresuspended in 100 μl of 0.5 N NaOH containing 0.5% ofpolyvinylpyrrolidone. 5 μl of lysate were mixed with 495 μl of a 20 mMTris-HCl solution, pH 8.0. For each experiment, two samples of 5 μl (forthe suspensions containing at least 1×10⁴ cfu.ml⁻¹) or three samples of5 μl (for the suspensions containing less than 1×10⁴ cfu.ml⁻¹) weretested in duplicate.

When the suspensions are mixed with onion seeds, the first step of theamplification makes it possible to detect 1×10⁶ cfu.ml⁻¹ (titerdetermined with the corresponding suspensions inoculated onto NCTM1medium). When the multiplex nested PCR is used, the limit of detectiongoes to approximately 1×10³ cfu.ml⁻¹, although a signal is frequentlyobtained (once or twice out of three tests) with suspensions containingapproximately 1×10² cfu.ml⁻¹. It should be noted that the equivalenttests carried out on the serial dilutions that were not mixed with onionseeds gave similar results.

These results show that it is possible to detect Xanthomonas axonopodispv. allii in a sample of seeds from a concentration of 10⁶ bacteria/mlafter the first amplification step, and from a concentration of 10³bacteria/ml or even 10² bacteria/ml after the second amplification step.

EXAMPLE 3 Detection of Xanthomonas axonopodis pv. allii on Infected SeedSamples

The presence of Xanthomonas axonopodis pv. allii was investigated in alot of onion seeds harvested on a contaminated experimental field(Humeau et al., Phytopathology, 96: 1345-1354, 2006). The number ofsamples for each experiment (35 per experiment), each consisting of 10 gof seeds, was determined according to a hypergeometric distribution inorder to detect at least one contaminated seed out of 30 000 seeds(r=0.05). Each experiment was carried out in duplicate. The seed sampleswere macerated in 50 ml of sterile 0.01 M Sigma 7-9 buffer (pH 7.2) for48 hours. The level of contamination of the lot was determined, firstly,by culturing, on NCTM1 medium, pure macerated materials or maceratedmaterials diluted to 1/10, and secondly, by means of at least twoanalyses of the macerated materials by multiplex nested PCRs afteralkaline extraction carried out as described above. Negative controls inwhich the sample was replaced with water were included in theexperiments.

In another series of experiments, the same lot of seeds was mixed withhealthy onion seeds in a 1:1.1, 1:2 and 1:12.8 proportion, and then thelevels of contamination of two, two and three independent samples ofthese mixtures were analyzed respectively by culturing on NCTM1 mediumand multiplex nested PCR under the conditions previously indicated.

Since the nested PCR amplification method is known to produce falsepositives, only the samples from which the expected DNA fragments weredetected at least twice are considered to be positive. In addition, thelevel of contamination determined from counting the dishes inoculatedand the analyses by multiplex nested PCR was calculated according to themethod described by Masmoudi et al. (Seed Sci. Technol.; 22: 407-414,1994). The correlation between the levels of contamination determined byculturing or by multiplex nested PCR and the ratio of the infectedseed/healthy seed dilutions was calculated by applying Pearson'scorrelation coefficient r (Pearson, K., Biometrika; 18: 105-117, 1926).

The results of these studies are recorded in table III below.

TABLE III Detection by Isolation on semi- multiplex nested PCR selectivemedium Number Number Number Number of lots of seed of positive ofpositive detected by the samples LC lots LC lots 2 methods Lot of seedsP2 Test 1 35 0.00007675 6 0.00004953 4 3 1/10.00)^(a) Test 2 350.0001213 9 0.00001183 1 1 mean 0.00010 0.00003068 Lot of seeds P2 Test1 35 0.00003658 3 0 0 0 mixed with healthy Test 2 35 0.00004953 40.00002402 2 0 seeds (1:1.1) mean 0.000043055 0.00001201 1/21.000^(b)Lot of seeds P2 Test 1 35 0.00003658 3 0 0 0 mixed with healthy Test 235 0.00003658 3 0.00001183 1 0 seeds (1:2) mean 0.00003658 0.0000059151/30000^(b) Lot of seeds P2 Test 1 35 0 0 0.00001183 1 0 mixed withhealthy Test 2 35 0 0 0 0 0 seeds (1:12.8) Test 3 35 0.00003658 30.00001183 1 0 1/138.600^(b) mean 1.21933E−03 0.0000079 Healthy seedsTest 1 35 0 0 0 0 Test 2 Test 2 35 0 0 0 0 ^(a)Levels of seedcontamination derived from the analyses by multiplex nested PCR^(b)Theoretical levels of seed contamination obtained by diluting theseed lot P2 with healthy seeds

The two methods of analysis made it possible to detect Xanthomonasaxonopodis pv. allii: six and nine positive samples were respectivelydemonstrated by multiplex nested PCR, which corresponds to a mean levelof contamination of 0.01%, whereas culturing on NCTM1 medium made itpossible to detect only three and four positive samples, whichcorresponds to a level of contamination of 0.0031%.

The series of experiments carried out with the seed lot mixed withhealthy onion seeds in a 1:1.1, 1:2 and 1:12.8 proportion, correspondingrespectively to theoretical contamination levels of 1/21 000, 1/30 000and 1/138 000, shows that the multiplex nested PCR method accuratelyestimates the levels of contamination: indeed, the positive correlationr between the theoretical level of contamination and the level ofcontamination determined experimentally by multiplex nested PCR is 0.90.Conversely, such correlation was not observed when the analysis wascarried out by culturing on NCTM1 medium.

At an experimentally determined level of contamination of 1/27 300, themultiplex nested PCR is positive in each test. For higher dilutions, inparticular 1/82 000, only one test out of three is positive.

Since the level of contamination recorded for epidemics of bacterialblight of onion in a tropical environment is 4.5/10 000, the detectionby multiplex nested PCR, with a sensitivity threshold of the method ofapproximately 1/27 300, is reliable and particularly suitable forcertifying Alliaceae seed lots.

1. An isolated polynucleotide comprising the polynucleotide sequence ofSEQ ID NO:
 6. 2. An isolated polynucleotide primer selected from thegroup consisting of: PXaa2U (SEQ ID NO: 7); PXaa2L (SEQ ID NO: 8);NXaa2U (SEQ ID NO: 9) and NXaa2L (SEQ ID NO: 10).
 3. A compositioncomprising the polynucleotide as claimed in claim 1, and/or of a pair ofprimers as claimed in claim 2, in an amount sufficient to detectXanthomonas axonopodis pv. allii.
 4. A method of screening forXanthomonas axonopodis pv. allii, which comprises: bringing DNA of abiological sample that may contain said bacterium into contact with oneor more polynucleotide(s) with the composition of claim 3 underconditions which allow selective hybridization between saidpolynucleotide(s) and the target sequence SEQ ID NO: 6, if said sequenceis present in said DNA; and detecting said hybridization.
 5. The methodas claimed in claim 4, comprising the following steps: (i) bringing theDNA of a biological sample to be tested into contact with a pair ofprimers selected from PXaa2U (SEQ ID NO: 7); PXaa2L (SEQ ID NO: 8);NXaa2U (SEQ ID NO: 9) and NXaa2L (SEQ ID NO: 10), under conditions whichallow selective hybridization between the primers and the targetsequence SEQ ID NO: 6; (ii) carrying out a polymerase chain reactionamplification under conditions which allow the amplification of thetarget sequence SEQ ID NO: 6; (iii) detecting the amplification product.6. The method as claimed in claim 5, further comprising a step (iv)which is a polymerase chain reaction amplification using a second pairof primers allowing the amplification of an internal fragment of theamplification product from step (ii).
 7. The method as claimed in claim4, characterized in that the DNA of the sample is also brought intocontact with at least one polynucleotide capable of selectivelyhybridizing, under stringent conditions, with the target sequence SEQ IDNO:
 5. 8. The method as claimed in claim 5, characterized in that, instep (i), the DNA of the sample is also brought into contact with one ormore polynucleotide(s) capable of selectively hybridizing, understringent conditions, with the target sequence SEQ ID NO: 5, and in thatthe conditions of step (ii) allow, in addition to the amplification ofthe target sequence SEQ ID NO: 6, the amplification of the targetsequence SEQ ID NO:
 5. 9. The method as claimed in claim 8,characterized in that the amplification step (iv) is carried out, inaddition, in the presence of a pair of primers allowing theamplification of an internal fragment of the amplification product fromthe first step of amplification of the polynucleotide of sequence SEQ IDNO:
 5. 10. The method as claimed in claim 9, characterized in that theamplification step (ii) is carried out with the pair of primers PXaa1U(SEQ ID NO: 1) and PXaa1L (SEQ ID NO: 2) and the pair of primers PXaa2U(SEQ ID NO: 7) and PXaa2L (SEQ ID NO: 8), and in that the amplificationstep (iv) is carried out with the pair of primers NXaa1U (SEQ ID NO: 3)and NXaa1L (SEQ ID NO: 4) and the pair of primers NXaa2U (SEQ ID NO: 9)and NXaa2L (SEQ ID NO: 10).
 11. The method as claimed in claim 4,characterized in that the biological sample is an Alliaceae seed.
 12. Akit which detects Xanthomonas axonopodis pv. allii, comprising thecomposition of claim
 3. 13. The detection kit as claimed in claim 12,further comprising one or more polynucleotide(s) capable of selectivelyhybridizing, under stringent conditions, with the target sequence SEQ IDNO: 5.